CN110643543A

CN110643543A - Preparation process of yellow wine rich in high-activity lactic acid bacteria

Info

Publication number: CN110643543A
Application number: CN201911042297.7A
Authority: CN
Inventors: 毛健; 史瑛; 刘双平; 韩笑
Original assignee: Jiangnan University (rugao) Food Biotechnology Research Institute; Rugao Jiangda Food Biotechnology Research Institute Co ltd; Jiangnan University
Current assignee: Jiangnan University (rugao) Food Biotechnology Research Institute; Rugao Jiangda Food Biotechnology Research Institute Co ltd; Jiangnan University
Priority date: 2019-10-30
Filing date: 2019-10-30
Publication date: 2020-01-03
Anticipated expiration: 2039-10-30
Also published as: CN110643543B

Abstract

The invention discloses a preparation process of yellow wine rich in high-activity lactic acid bacteria, and belongs to the technical field of biology. The invention provides a strain of lactobacillus hilgardii 5-1 capable of tolerating yellow wine, and the lactobacillus hilgardii 5-1 is mixed by 3 multiplied by 10⁹Adding into yellow wine at a concentration of CFU/mL, storing at 4 deg.C, and after 30 days, the viable count of Lactobacillus hilgardii 5-1 in yellow wine can still be as high as 1 × 10⁶～1×10⁷CFU/mL, the Lactobacillus hilgardii 5-1 was added at 3X 10⁹The CFU/mL concentration is added into yellow wine and stored at 37 deg.C, and after 24 hr, viable count of Lactobacillus hilgardii 5-1 in yellow wine can still reach (1.55 + -0.03) x 10⁹CFU/mL, therefore, the Lactobacillus hilgardii 5-1 has extremely high application prospect in preparing yellow wine rich in active lactobacillus.

Description

Preparation process of yellow wine rich in high-activity lactic acid bacteria

Technical Field

The invention relates to a preparation process of yellow wine rich in high-activity lactic acid bacteria, belonging to the technical field of biology.

Background

The yellow wine is a fermented wine with low alcoholic strength prepared by using grains (glutinous rice, husked millet and corn) as raw materials, wheat starter as a saccharifying agent and yellow wine yeast or active dry yeast as a fermenting agent through the steps of rice soaking, cooking, saccharifying, fermenting, squeezing, filtering, wine decocting, storing and blending. It has a long history, and is called three ancient wines in the world together with beer and wine. The yellow wine is low-alcoholicity wine, and retains nutrition and active substances generated during fermentation. Therefore, yellow wine contains a plurality of functional substances beneficial to human health, such as proteins, inorganic salts, trace elements, vitamins, functional oligosaccharides and polysaccharides, polyphenol substances, inhibitory neurotransmitter gamma-aminobutyric acid, bioactive peptides and the like.

Along with the development of society, people have higher and higher pursuits for health, and the concept that strong liquor is harmful to human bodies is also deeply fixed in the brains of people, so that the attraction of yellow wine with low alcohol content, nutrition and relative health to liquor enthusiasts is gradually increased, and the rapid development of the yellow wine industry is promoted. Therefore, increasing the probiotic performance of yellow wine is more important for yellow wine brewing manufacturers who want to increase the market competitiveness of yellow wine.

Lactic acid bacteria are a probiotic bacterium that can ferment carbohydrates to lactic acid. A large number of researches show that the lactobacillus can promote the growth of animals, improve the gastrointestinal function, improve the digestibility and the biological value of food, reduce serum cholesterol, control endotoxin, inhibit the growth of putrefying bacteria in intestinal tracts, improve the immunity of organisms and the like. Therefore, the yellow wine rich in active lactobacillus is very helpful for increasing the probiotic performance of the yellow wine product.

However, yellow wine is rich in some substances that inhibit the survival or growth of lactic acid bacteria, such as ethanol, acetic acid, and higher alcohols, which makes it difficult for lactic acid bacteria to survive in yellow wine for a long time. Therefore, a lactic acid bacterium which can tolerate yellow wine and can survive in the yellow wine for a long time is urgently needed to be found, so that the preparation of the yellow wine rich in the active lactic acid bacterium becomes possible.

Disclosure of Invention

[ problem ] to

The technical problem to be solved by the invention is to provide a strain of Lactobacillus hilgardii (Lactobacillus hilgardii) which can tolerate yellow wine.

[ solution ]

In order to solve the technical problems, the invention provides a Lactobacillus hilgardii strain 5-1, wherein the Lactobacillus hilgardii strain 5-1 is preserved in the China center for type culture collection in 2019, 07-25.M.in Wuhan, Wuhan university with the preservation number of CCTCC NO: M2019583.

The Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 is derived from a brewed yellow wine sample in the Shaoxing region (a sample obtained in the yellow wine fermentation process), the strain is subjected to sequencing analysis, the 16S rDNA sequence of the strain is shown as SEQ ID NO.1, the sequence obtained by sequencing is subjected to nucleic acid sequence alignment in NCBI, and the result shows that the similarity of the nucleic acid sequence with the Lactobacillus hilgardii is 99.04%, so that the strain is named as Lactobacillus hilgardii (Lactobacillus hilgardii).

The colony of the Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 on the MRS culture medium is round, milky white, convex and neat in edge.

The invention also provides application of the Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 in wine making.

In one embodiment of the present invention, the viable bacteria content of Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 is not less than 1 × 10⁶CFU/mL。

In one embodiment of the present invention, the viable bacteria content of the above Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 in the wine is 1X 10⁸～1×10⁹CFU/mL。

In one embodiment of the invention, the wine is yellow wine.

The invention also provides a wine, which contains the Lactobacillus hilgardii (Lactobacillus hilgardii) 5-1.

In one embodiment of the invention, the wine is yellow wine.

The invention also provides a method for preparing the wine, which is characterized in that the method is to add the Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 into the wine to obtain the wine.

In one embodiment of the present invention, the method comprises inoculating the Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 into a culture medium to culture, and obtaining a culture solution; centrifuging the culture solution, and collecting thalli; adding the thallus into wine, and resuspending to obtain the above wine.

In one embodiment of the present invention, the method comprises inoculating the Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 into a culture medium to culture, and obtaining a culture solution; centrifuging the culture solution, and collecting thalli; and washing the thalli for 2-3 times by using normal saline or buffer solution, adding the thalli into wine, and carrying out heavy suspension to obtain the wine.

In one embodiment of the present invention, the medium is MRS medium, MC medium or LBS medium.

In one embodiment of the present invention, the temperature of the culture is 35 to 38 ℃.

In one embodiment of the invention, the buffer is PBS buffer.

In one embodiment of the invention, the wine is yellow wine.

[ advantageous effects ]

The invention provides a strain of lactobacillus hilgardii (Lactob) capable of tolerating yellow wineBacillus hilgardii)5-1, and culturing the Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 at a temperature of 3X 10⁹The CFU/mL concentration is added into yellow wine and stored at 4 deg.C, after 30 days, the viable count of Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 in yellow wine can still be as high as 1 × 10⁶～1×10⁷CFU/mL, the Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 was added at 3X 10⁹The CFU/mL concentration is added into yellow wine and stored at 37 deg.C, and after 24 hr, the viable count of Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 in yellow wine can still be as high as (1.55 + -0.03) × 10⁹CFU/mL, therefore, the Lactobacillus hilgardii 5-1 has extremely high application prospect in preparing yellow wine rich in active Lactobacillus.

Biological material preservation

The Lactobacillus hilgardii strain 5-1 is characterized in that the Lactobacillus hilgardii strain 5-1 is preserved in the China center for type culture collection in 2019 at the number of CCTCC NO: M2019583 and the preservation address is Wuhan university, Wuhan China.

Drawings

FIG. 1: morphological pictures of Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 were microscopically examined.

FIG. 2: 5-1 of Lactobacillus hilgardii, n3 of fructo-eating Lactobacillus (Lactobacillus fructivorans) and 19-4 of Lactobacillus fermentum, wherein the viable count of the Lactobacillus hilgardii, the Lactobacillus fructivorans and the Lactobacillus fermentum are preserved in yellow wine for 3d, 7d, 10d, 15d, 20d and 30d at 4 ℃.

FIG. 3: the viable count of Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 after 3d, 7d, 10d, 15d, 20d and 30d of storage in yellow wine at 25 ℃ or 4 ℃ respectively.

Detailed Description

The invention is further elucidated with reference to a specific embodiment and a drawing.

The yellow rice wine related in the following formula embodiment is purchased from Zhejiang Guyue Longshan Shaoxing wine, Huizshan Shaoxing wine, and Shanghai Jinfeng wine, respectively, and the alcoholic strength of the yellow rice wine purchased from Zhejiang Guyue Longshan Shaoxing wine, Huizshan Shaoxing wine, and Shanghai Jinfeng wine, respectively is 8-12% vol, and the total acid content is 3.5-8.0 g/L (calculated by lactic acid).

The media involved in the following examples are as follows:

MRS solid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate₂HPO₄·3H₂O 2.6g/L、MgSO₄·7H₂O 0.1g/L、MnSO₄·H₂O0.05 g/L, Tween 801mL/L, agar 15g/L, cysteine hydrochloride 0.5 g/L.

MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate₂HPO₄·3H₂O 2.6g/L、MgSO₄·7H₂O 0.1g/L、MnSO₄·H₂O0.05 g/L, Tween 801mL/L and cysteine hydrochloride 0.5 g/L.

Example 1: screening of lactic acid bacteria and identification of the bacteria

1. Screening

Pretreating a brewed yellow wine sample (a sample obtained in the yellow wine fermentation process) from Shaoxing region, storing the sample in a refrigerator at-80 ℃ in 20% glycerol, taking out and thawing the sample, sucking 0.5mL of the sample into 4.5mL of the sample, performing gradient dilution by using 0.9% (m/m) physiological saline containing 0.05g/L cysteine, selecting a proper gradient dilution solution to coat the sample on an MRS solid culture medium containing 10% (v/v) ethanol, culturing the sample at 37 ℃ for 48h, picking a relatively large colony to transfer the colony to an MRS liquid culture medium containing 10% (v/v) ethanol, culturing the colony at 37 ℃ for 48h, and detecting the OD (optical density) of the culture medium by using an ultraviolet spectrophotometer₆₀₀Selecting OD₆₀₀Diluting the high culture solution, coating the diluted high culture solution on an MRS solid culture medium, culturing for 48h at 37 ℃, selecting a typical bacterial colony of lactic acid bacteria, continuously inoculating the typical bacterial colony on the MRS solid culture medium, streaking and purifying, selecting a single bacterial colony, transferring the single bacterial colony to the MRS liquid culture medium for enrichment, and preserving by 30% glycerol to obtain a bacterial strain A, B, C;

the bacterial colony of the strain A is circular, milky white, convex and neat in edge, the bacterial colony of the strain B is circular and whitish, and the bacterial colony of the strain C is circular, milky white and slightly transparent and is a typical bacterial colony of lactic acid bacteria.

2. Identification

Extracting genomes of the strains A-C, amplifying and sequencing the 16S rDNA of the strains A-C (wherein the nucleotide sequence of the 16S rDNA obtained by amplifying the strain A is shown as SEQ ID NO. 1), and comparing the obtained sequences in NCBI (national center for Biotechnology information) to obtain a sequence shown in the following result: the strain A is Lactobacillus hilgardii which is named as Lactobacillus hilgardii (Lactobacillus hilgardii) 5-1; the strain B is Lactobacillus fructosanus, named Lactobacillus fructicola (Lactobacillus fructivorans) n 3; the strain C is lactobacillus fermentum and is named as lactobacillus fermentum (Lactobacillus) 19-4.

Example 2: culture and Observation of lactic acid bacteria

Inoculating Lactobacillus hilgardii (Lactobacillus hilgardii)5-1, Lactobacillus fructosus (Lactobacillus fructivorans) n3 and Lactobacillus fermentum (Lactobacillus fermentum)19-4 into MRS solid culture medium, anaerobically culturing at 37 deg.C for 24 hr, transferring into fresh MRS liquid culture medium, culturing under the same conditions for 24 hr, centrifuging thallus 6000g for 15min, washing thallus with 0.9% physiological saline solution, centrifuging 6000g again for 10min to obtain Lactobacillus hilgardii (Lactobacillus hilgardii)5-1, Lactobacillus fructosus (Lactobacillus fructivorans) n3 and Lactobacillus fermentum (Lactobacillus fermentum)19-4 thallus.

The bacterial cells of Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 were observed under a microscope (see FIG. 1 for observation results), and were found to be rod-shaped.

Example 3: tolerance of different lactic acid bacteria to yellow wine

Adding 5-1 of Lactobacillus hilgardii (Lactobacillus hilgardii), 5-1 of Lactobacillus fructosus (Lactobacillus fructivorans) n3 and 19-4 of Lactobacillus fermentum (Lactobacillus fermentum) into yellow wine (obtained from Shang-Yue-Longshan Shaoxing wine, Inc., Huizhan Shaoxing wine, Inc., and Shanghai Jinfeng wine, Inc., respectively) 5-1 of Lactobacillus hilgardii (Lactobacillus hilgardii), and Lactobacillus fructosus (Lactobacillus fr) 5-1The viable count of the eutivorans) n3 and the viable count of the lactobacillus fermentum (Lactobacillus) 19-4 are both 3 multiplied by 10⁹CFU/mL to obtain yellow wine A1, B1 and C1 rich in lactobacillus; the yellow wine A1, B1 and C1 rich in lactic acid bacteria are stored at 37 ℃ for 24 hours, and after the yellow wine A1, B1 and C1 rich in lactic acid bacteria, the viable count of Lactobacillus hilgardii (Lactobacillus hilgardii)5-1, Lactobacillus fructovorans (Lactobacillus fructivorans) n3 and Lactobacillus fermentum (Lactobacillus fermentum)19-4 is measured by a plate counting method after the yellow wine A1, B1 and C1 are stored for 24 hours, and the detection result is shown in Table 1.

Respectively adding 5-1 of Lactobacillus hilgardii (Lactobacillus hilgardii), n3 of Lactobacillus fructovorus (Lactobacillus fructivorans) and 19-4 of Lactobacillus fermentum (Lactobacillus fermentum) into yellow wine (the yellow wine is obtained from Shang-Yue-Longshan Shaoxing wine, Inc., Huizhan Shaoxing wine, Inc., and Shanghai Jinfeng wine, Inc., wherein the viable count of the Lactobacillus hilgardii (Lactobacillus hilgardii)5-1, the viable count of the Lactobacillus fructovorus (Lactobacillus fructivorans) n3 and the viable count of the Lactobacillus fermentum 19-4 are all 3 × 10⁹CFU/mL to obtain yellow wine A2, B2 and C2 rich in lactobacillus; the yellow wine A2, B2 and C2 rich in lactic acid bacteria are stored at 4 ℃ for 30 days, and the viable count of Lactobacillus hilgardii (Lactobacillus hilgardii)5-1, Lactobacillus fructovorans (Lactobacillus fructivorans) n3 and Lactobacillus fermentum (Lactobacillus fermentum)19-4 in the yellow wine A2, B2 and C2 rich in lactic acid bacteria is determined by a plate counting method when the yellow wine A2, B2 and C2 rich in lactic acid bacteria are stored for 3 days, 7 days, 10 days, 15 days, 20 days and 30 days respectively, and the detection result is shown in figure 2.

As is clear from Table 1, the numbers of viable Lactobacillus hilgardii (Lactobacillus hilgardii)5-1, Lactobacillus fructovorans (Lactobacillus fructivorans) n3 and Lactobacillus fermentum (Lactobacillus fermentum)19-4 were all high in yellow wine containing Lactobacillus hilgardii (Lactobacillus hilgardii)5-1, Lactobacillus fructivorans (Lactobacillus fructivorans) n3 and Lactobacillus fermentum (Lactobacillus fermentum)19-4, respectively, after storing the yellow wine at 37 ℃ for 24 hours.

However, as is clear from FIG. 2, when Lactobacillus fructosus (Lactobacillus fructivorans) n3 and Lactobacillus fermentum (Lactobacillus fermentum)19-4 were stored in yellow wine at 4 ℃ for only 10 days, the viable count of Lactobacillus fructosus (Lactobacillus fructivorans) n3 and Lactobacillus fermentum (Lactobacillus fermentum)19-4 in yellow wine containing Lactobacillus fructosus (Lactobacillus fructivorans) n3 and Lactobacillus fermentum (Lactobacillus fermentum)19-4, respectively, was almost reduced to 0; when the Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 is stored in yellow wine at 4 ℃, after 10-30 days of storage, the viable count of the Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 in the yellow wine containing the Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 can still reach 1 × 10⁶～1×10⁷CFU/mL。

It can be seen that only Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 has strong resistance to yellow wine, and Lactobacillus fructovorans (Lactobacillus fructivorans) n3 and Lactobacillus fermentum (Lactobacillus fermentum)19-4 do not have such properties.

TABLE 1 viable count of different lactic acid bacteria after 24h storage in yellow wine at 37 deg.C

Bacterial strains	Viable count (CFU/mL)
		Lactobacillus hilgardii 5-1	(1.55±0.03)×10⁹
Lactobacillus fermentum 19-4	(2.84±0.17)×10⁹
		Lactobacillus fructosanus n3	(2.15±0.54)×10⁹

Example 4: tolerance of Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 to yellow wine at different storage temperatures

Adding Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 into yellow wine (the yellow wine is obtained from Zhejiang Guyue Longshan Shaoxing wine, Huizhan Shaoxing wine, and Shanghai Jinfeng wine, respectively), wherein the viable count of Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 is 3 × 10⁹CFU/mL to obtain yellow wine rich in lactobacillus; half of the yellow wine rich in Lactobacillus is stored at 4 ℃ for 30 days, the other half of the yellow wine rich in Lactobacillus is stored at 25 ℃ for 30 days, the viable count of Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 in the two parts of yellow wine rich in Lactobacillus is determined by a plate counting method after 3 days, 7 days, 10 days, 15 days, 20 days and 30 days are respectively stored, and the detection result is shown in figure 3.

As can be seen from FIG. 3, the normal temperature environment of 25 ℃ is not favorable for the survival of Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 in yellow wine, and the yellow wine containing Lactobacillus hilgardii (Lactobacillus hilgardii)5-1 should be refrigerated at a low temperature of, for example, 4 ℃.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> university of south of the Yangtze river

<120> preparation process of yellow wine rich in high-activity lactic acid bacteria

<160> 1

<170> PatentIn version 3.3

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<211> 1152

<212> DNA

<213> Lactobacillus hilgardii (Lactobacillus hilgardii)

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gttgtaccgt ccattgtagc acgtgtgtag cccaggtcat aaggggcatg atgatttgac 300

gtcatcccca ccttcctccg gtttgtcacc ggcagtctcg ccagagtgcc caactaaatg 360

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taggttagca gaagatgtca agacctggta aggttcttcg cgtagcatcg aattaaacca 540

catgctccac cgcttgtgcg ggcccccgtc aattcctttg agtttcaacc ttgcggtcgt 600

actccccagg cggagtgctt aatgcgttag ctgcagcact gaagggcgga aaccctccaa 660

cacttagcac tcatcgttta cggcatggac taccagggta tctaatcctg ttcgctaccc 720

atgctttcga gcctcagcgt cagttgcaga ccagacagcc gccttcgcca ctggtgttct 780

tccatatatc tacgcatttc accgctacac atggagttcc actgtcctct tctgcactca 840

agtctcccgg tttccgatgc acttctccgg ttaagccgaa agctttcaca tcagacctaa 900

aaaaccgcct gcgctcgctt tacgcccaat aaatccggac aacgcttgcc acctacgtat 960

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Claims

1. A Lactobacillus hilgardii strain is characterized in that the Lactobacillus hilgardii strain is preserved in China center for type culture collection (CCTCC NO: M2019583) in 2019, 07-25 months.

2. Use of Lactobacillus hilgardii (Lactobacillus hilgardii) according to claim 1 for the preparation of wine.

3. A liquor comprising the Lactobacillus hilgardii strain of claim 1.

4. A wine according to claim 3 wherein the Lactobacillus hilgardii (Lactobacillus hilgardii) according to claim 1 has a viable bacteria content of not less than 1 x 10⁶CFU/mL。

5. A wine as claimed in claim 3 or claim 4 which is yellow wine.

6. A process for the preparation of a wine according to any one of claims 3 to 5, wherein the process comprises adding Lactobacillus hilgardii (Lactobacillus hilgardii) according to claim 1 to a wine to obtain a wine according to any one of claims 3 to 5.

7. The method according to claim 6, wherein the Lactobacillus hilgardii (Lactobacillus hilgardii) according to claim 1 is inoculated into a culture medium and cultured to obtain a culture solution; centrifuging the culture solution, and collecting thalli; adding the thallus into wine, and resuspending to obtain the wine of any one of claims 3-5.

8. The method according to claim 6 or 7, wherein the Lactobacillus hilgardii (Lactobacillus hilgardii) according to claim 1 is inoculated into a culture medium and cultured to obtain a culture solution; centrifuging the culture solution, and collecting thalli; washing the thallus with normal saline or buffer solution for 2-3 times, adding into wine, and resuspending to obtain the wine of any one of claims 3-5.

9. The method according to any one of claims 6 to 8, wherein the medium is MRS medium, MC medium or LBS medium.

10. The method according to any one of claims 6 to 9, wherein the temperature of the culture is 35 to 38 ℃.